Where the term “mass” is used below it does not refer to the “physical mass” m, but to the “charge-related mass” m/z, where z is the number of the ion's uncompensated elementary charges. When simply “mass” or the “mass of the ions” is referred to here, it is always to be understood as the charge-related mass fraction m/z unless there is a note expressly stating otherwise. The terms “light ions” and “heavy ions” also relate to the charge-related mass fraction m/z.
The investigation of the structures, characteristics and activities of proteins, and of other biopolymers as well, is largely based on so-called “tandem mass spectrometry”, which not only provides spectra of the mixtures of protein ions, but also allows individual types of protein ions to be exposed to certain reactions and their reaction products to be analyzed. A particularly interesting and frequently used type of such reactions is fragmentation, where “parent ions” are first selected for a fragmentation and then fragmented to form “daughter ions” so that the daughter ions produced can be measured in a mass spectrum. These daughter ion mass spectra contain information on the primary and secondary structures of the proteins that not only allows the genetically determined basic structure of their amino acids (the “sequence”) to be identified, but also enables the recognition of type and localization of modifications that are important because they often cause a change of function (“post translational modifications”, PTM).
Tandem mass spectrometry includes the following three individual steps: (1) selection of the analyte ions to be analyzed; (2) reactions causing a change; (3) mass analysis of the reaction products. These steps can be carried out sequentially in the same storage unit (“tandem-in-time”) using storage mass spectrometers such as ion traps. However, it is also possible to spread these steps over three parts of the instrument (“tandem-in-space”): (1) selection of the analyte ions to be analyzed with a first mass analyzer, the “mass selector”; (2) reactions in a special reaction cell; and (3) mass analysis of the reaction products in a second mass analyzer, where mass analyzers with very high mass resolution can be used. From a historical point of view, tandem mass spectrometry in space, spread over different parts of the instrument, is older.
If the emphasis is on fast scanning rate, high mass resolution and high mass accuracy, the reaction products generated are preferably measured in time-of-flight mass spectrometers with injection of the ions orthogonal to the flight tube (OTOF-MS). Only if the measurement speed is of entirely secondary importance, modern embodiments of Kingdon ion traps or ion cyclotron resonance mass spectrometers can be used as mass analyzers, offering high mass resolution. However, these Fourier transform mass spectrometers have a slow scanning rates.
For the fragmentation of proteins or similar biopolymers there are essentially only two fundamentally different types of fragmentation available, “ergodic” and “non-ergodic” or “electron-induced” fragmentation, for each of which many different favorable embodiments exist. The ergodic fragmentations include collision-induced fragmentation of ions by collisions with the molecules of a damping or collision gas often referred to as collision-induced dissociation (CID), a method which has the shortcoming of a small mass range, and has difficulties with the fragmentation of heavy ions, however. A much more suitable method is ion-collision-induced fragmentation. An outstanding method of electron-induced fragmentation is “electron transfer dissociation” (ETD), a fragmenting reaction between ions with multiply positive and suitably negative charges.
The two types of fragmentation, ergodic and electron-induced, lead to two significantly different kinds of fragment ion spectra whose information contents are complementary and that deliver particularly detailed information on the structures of the analyte ions when both types of fragment ion spectra are measured. As the specialist is aware, the fragment ions of electron-induced fragmentations belong to the so-called c and z series and are therefore very different to the fragment ions of the b and y series, which are obtained by ergodic fragmentations. In particular, with electron transfer dissociation all side chains are preserved, which are lost with ergodic fragmentation, among them the important post translational modifications such as phosphorylations, sulfations and glycosylations. But for other analyses as well, for example de-novo sequencing, it is advantageous or even absolutely imperative to compare fragment ion spectra of good quality which have been obtained ergodically and by electron-induced methods.
The invention presented here refers in particular to electron transfer dissociation and the reaction cell required here, although it should also be possible to carry out ergodic fragmentations, such as collision-induced fragmentations, in the same reaction cell. Both types of fragment ion spectra should satisfy the highest quality requirements. A modem tandem mass spectrometer for bioanalysis must provide both types of fragmentation in methods with as few shortcomings as possible.
Electron transfer dissociation can be carried out in ion traps in which positive and also negative ions can be stored and can react with each other by introducing suitable negative ions to the stored positive analyte ions. Methods of this type have been described in U.S. Pat. No. 7,456,397 B2 and U.S. Published Application 2005/0199804 A1.
The fragmentation of protein ions by electron transfer is brought about by reactions between multiple positively charged protein ions and suitable negative reactant ions. Suitable negative reactant ions are usually specially selected radical anions, such as those of fluoranthene, fluorenone, anthracene or other polyaromatic compounds, but also of azulene or of non-aromatic compounds such as 1-3-5-7-cyclooctatetraene, for example. These radical anions can react very easily to form a stable neutral molecule with a complete electron configuration by releasing electrons. As described in the two patent documents cited above, they are generated, for example, in negative chemical ionization ion sources (NCI) by single electron capture or by electron transfer. The NCI ion sources are also called electron attachment ion sources.
The radical anions of suitable substances can also be produced directly or indirectly in electro-spray ion sources as are usually found in time-of-flight mass spectrometers with orthogonal ion injection. Indirect generation means that anions of certain substances are generated first and then converted into the radical anions which are useful as the reactant ions for ETD by careful collision-induced dissociation (“Electron-Transfer Reagent Anion Formation via Electrospray Ionization and Collision-Induced Dissociation”, T.-Y. Huang et al., Anal. Chem. 2006, 78, 7387-7391).
So far, linear ion traps (“2D ion traps”) have exclusively been used as ETD fragmentation cells in tandem mass spectrometers with high-resolution mass analyzers. Although ETD fragmentation can also be performed advantageously in three-dimensional ion traps (“3D ion traps”), the commercial embodiments of the 3D ion traps used have so far been limited to those mass spectrometers that use this 3D ion trap simultaneously and exclusively as a mass analyzer for measuring the fragmentation ion spectra. They are not designed to transfer the fragment ions into a different mass analyzer, and this is only possible with some effort and expense. In U.S. Published Patent Application 2009/0283675 A1 a 3D ion trap is proposed which is configured to transfer the fragment ions to a mass analyzer of higher mass resolution. The advantage of such a configuration is the high ETD fragmentation yield, for which there is also a hypothetical explanation in the document; a disadvantage is that it is more difficult to fill the 3D ion trap than a 2D ion trap.
For fragmentation by electron transfer, at least doubly, preferably triply, quadruply or even higher charged parent ions are selected; in some cases parent ions with a 10- or even 15-fold charge are fragmented. In linear ion traps, the freshly introduced parent ions are stored in the axis of the rod system in the form of a string-like cloud with small diameter after their kinetic energy has been damped by a collision gas. The linear ion traps are usually designed as multipole rod systems, i.e., as quadrupole, hexapole or octopole rod systems with two, three or four pairs of pole rods. A hexapole rod system is depicted in FIG. 1. The two RF voltage phases of opposite polarity are applied to the pole rods alternately around the circumference and generate a radially repulsive pseudopotential in the interior.
Quadrupole rod systems display a quadratic increase in the pseudopotential in the radial direction; the radial oscillations of the (undamped) ions are harmonic. Under the influence of a damping gas, with which these rod systems are usually filled, the ions collect in only a few milliseconds as a string-shaped cloud in the axis of the rod system. Quadrupole rod systems are not usually used as collision cells for fragmentations; more common are hexapole rod systems, which have a cubic pseudopotential increase and thus the pseudopotential well in the axis has a shallower bottom. The string-shaped cloud has a slightly larger diameter due to the smaller retroactive force.
A “pseudopotential” is not a real potential, but describes only the time-averaged action of the force of an inhomogeneous RF field on ions of arbitrary polarity. An RF voltage which is applied to the tip of an electrode, a wire or a pole rod generates such a repulsive inhomogeneous electric field. An RF dipole field also generates a pseudopotential that drives ions away from the dipole. Ions on the axis of the dipole are driven to the center of the dipole.
In this document all systems which confine ions radially with the aid of pseudopotentials, particularly the multipole rod systems, are called “ion guides”, because they can transmit the ions in their interior. In this sense, the linear ion traps, and also quadrupole mass filters or the so-called ion funnels are ion guides, even if this is not their primary purpose.
Commercial instruments have so far used methods that trap positive and negative ions simultaneously in a reaction cell to induce unhindered reactions between the two species of ions in a controllable way. The principle for this storage is disclosed in U.S. Pat. No. 5,572,035 entitled “Method and Device for the Reflection of Charged Particles on Surfaces” to J. Franzen. This patent states all types of cylindrical or conical ion guides can be used as storage devices if the end openings are barred for the exit of ions by reflecting rf or dc potentials. It also states that with rf field reflection, ions of both polarities can be stored. In addition, it states with dc potentials, ion guides store ions of a single polarity only. This patent specification is concerned in a very general way with the reflection of ions of both polarities at pseudopotentials which are formed by inhomogeneous RF fields.
The confinement of ions in linear ion traps, which in the sense of this document belong to the ion guides, by an RF-generated pseudopotential barrier is therefore known. There are a variety of embodiments, however. The review article by Y. Xia and S. A. McLuckey provides an overview: “Evolution of Instrumentation for the Study of Gas-Phase Ion/Ion Chemistry via Mass Spectrometry”, J Am Soc Mass Spectrom 2008, 19, 173-189.
Another embodiment is described in U.S. Pat. No. 7,026,613 B2 to J. Syka. In this specification, periodic voltages are applied to a first set of electrodes to generate a first oscillating electric potential, which confines the ions radially in an ion channel, and periodic voltages are applied to a second set of electrodes in order to generate a second oscillating electric potential that confines the ions axially in the ion channel. (Citation from the Summary: “Periodic voltages are applied to electrodes in the first set of electrodes to generate a first oscillating electric potential that radially confines the ions in the ion channel, and periodic voltages are applied to electrodes in the second set of electrodes to generate a second oscillating electric potential that axially confines the ions in the ion channel”) The complex wording states nothing more than that a radially confining ion guide (the first set of electrodes) is closed for ions of both polarities with the aid of RF voltages at a second set of electrodes (which are necessarily arranged separately in the axial direction) by erecting a pseudopotential barrier in the axial direction. The essence of the disclosure in U.S. Pat. No. 7,026,613 is therefore solely the introduction of electrodes for the axially terminating RF voltage, which are not expressly mentioned in U.S. Pat. No. 5,572,035 by J. Franzen, but are of course inherently necessary.
It must be expressly pointed out here that the two RF voltages disclosed in U.S. Pat. No. 7,026,613 are applied to two different sets of electrodes. This leads to pseudopotential barriers with an unfavorable form, however. If the second RF voltage is applied to terminating electrodes at the end of the pole rods, for example to apertured diaphragms, a pseudopotential is generated with two maxima (see FIG. 5 top). An apertured diaphragm acts like the ring electrode of a three-dimensional ion trap and creates a storage space in the form of a pseudopotential well in the plane of the apertured diaphragm. The potential well of the storage space is terminated by a barrier at each end of the pseudopotential. If these double barriers are switched on by the RF voltage at the apertured diaphragm, filling is difficult because some of the ions always remain in the potential well of the storage space of the apertured diaphragm. Ion traps with this technology are therefore only ever filled with the pseudopotential barriers switched off, but this requires specially designed ion traps to solve the problems which then occur. In a commercially available instrument, where ETD is carried out in a linear ion trap of this type, the ion trap used is therefore segmented into three sections. The axis potentials of the sections can be set separately. This makes it possible to introduce positive and negative ions successively and store them in the interim in different sections of the linear ion trap, before the ions are mixed by equalizing the axis potentials and the reactions thus started.
U.S. Pat. No. 7,227,130 B2 entitled “Method for Providing Barrier Fields at the Entrance and Exit End of a Mass Spectrometer” (J. W. Hager and F. A. Londry, 2005) discloses that auxiliary RF voltages at the entrance and exit are applied to the terminating electrodes for a linear ion trap formed from long pole rods in order to trap ions of both polarities in the axial direction, the auxiliary RF voltages being obtained from the main RF voltage at the pole rods by voltage dividers. This is a special embodiment of the system disclosed in U.S. Pat. No. 7,026,613.
U.S. Pat. No. 7,288,761 B2 (B. A. Collings, 2005) describes for the first time the possibility of not applying the axially confining RF voltage to electrodes at the end of a multipole rod system, but making the axis potential of the rod system oscillate at RF with reference to its environment. This generates axial pseudopotential barriers at the ends of the multipole rod system. This method requires only one RF generator. The oscillating axis potential is generated either by an asymmetric arrangement of the pole rods about the axis or by two different amplitudes for the two phases of the RF voltage. Despite the advantageous use of only one RF generator there is the disadvantage here that the amplitude of the oscillating axis potential cannot easily be adjusted to the mass range of the ions to be trapped, because this requires that either the separations between the pole rods or the transformer for the generation of the two amplitudes must be changed for one of the two phases of the RF voltage.
PCT Application WO 2009 006726 (I. Chernushevich and A. Loboda, 2007) and German Patent Application DE 10 2008 055 899.0 (C. Stoermer, 2008) disclose operating mode for linear RF ion traps with a new type of electrical configuration for the pole rods. Multipole rod systems are used as RF ion traps. The configuration uses two RF voltages whose amplitudes can be set individually. The two phases of a first two-phase RF voltage are applied to the pole rods as usual, i.e., alternately around the circumference; this traps ions of both polarities radially. The second single-phase RF voltage is applied to all pole rods simultaneously; it produces practically no field in the interior of the rod system, but a single pseudopotential barrier at each end (see FIG. 5 bottom), which axially traps ions of both polarities. The ion trap can thus store positive and negative ions simultaneously without the need to apply an RF voltage to the terminating electrodes. The amplitude of the axially confining barrier of the pseudopotential can be adjusted independently of the radially confining pseudopotential. This arrangement represents the closest prior art for the invention presented here.
Decisive for assessing the quality of a reaction cell is the yield of product ions; for fragmentation by electron transfer (ETD) this means the yield of fragment ions. Here, all linear ion traps so far have had considerable disadvantages compared to the three-dimensional ion traps. A possible cause for this is listed in the German Patent Application DE 10 2008 023 693 A1 already mentioned above.
An objective of the invention is to provide a reaction cell which offers a high fragmentation yield for ETD fragmentation, but which is easier to fill with ions than a 3D ion trap. It shall also be suitable for collision-induced fragmentations of different types.